Various publications, including patents, published applications, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference herein, in its entirety and for all purposes.
Protein kinases are among the important classes of therapeutic targets because of their central roles in cell signaling pathways. The presence of a highly conserved ATP-binding site in kinases consists of a deep hydrophobic pocket, adapted for small molecule binding, that can be exploited by agents. Due to the evolutionary conservation of this pocket between protein kinases, however, achieving highly selective kinase inhibition by ATP-competitive inhibitors is a significant challenge. In drugs, these off-target activities can produce dose-limiting toxicities that limit therapeutic efficacy. In research compounds, off-target activities confound experimental interpretation. Thus, defining the specificity of kinase inhibitors using broad panels of diverse protein kinases is important.
Knowledge of target selectivity for kinase inhibitors is important for predicting and interpreting the effects of inhibitors in both the research and clinical settings. Recent technological advances have led to the development of several methods to profile kinase target selectivity against significant fractions of the 518 human protein kinases. These include kinase-inhibitor binding (or displacement) assays, cell-based profiling methods, and high-throughput enzymatic assays. Initial applications of these methods have revealed a striking degree of promiscuity of these compounds, even those thought highly specific. Off-target inhibition is frequently observed even of kinases only distantly related to the primary target. These findings have emphasized the importance of comprehensive testing of kinase inhibitor specificity.
Generally, kinase inhibitors have been identified in a target-centric manner in which inhibitors are developed through an iterative process against a particular kinase of interest. The resulting compounds are then tested for specificity against a panel of representative kinases. An alternative approach has been suggested in which large libraries of compounds are initially screened in parallel against comprehensive panels of recombinant protein kinases. Compounds showing desired selectivity patterns are then chemically optimized for the desired target(s). The cost of implementing this strategy for very large compound libraries, however, is prohibitive.